Abstract
The UKMYC5 plate is a 96-well microtiter plate designed by the CRyPTIC Consortium (Comprehensive Resistance Prediction for Tuberculosis: an International Consortium) to enable the measurement of MICs of 14 different antituberculosis (anti-TB) compounds for >30,000 clinical Mycobacterium tuberculosis isolates. Unlike the MYCOTB plate, on which the UKMYC5 plate is based, the UKMYC5 plate includes two new (bedaquiline and delamanid) and two repurposed (clofazimine and linezolid) compounds. UKMYC5 plates were tested by seven laboratories on four continents by use of a panel of 19 external quality assessment (EQA) strains, including H37Rv. To assess the optimal combination of reading method and incubation time, MICs were measured from each plate by two readers, using three methods (mirrored box, microscope, and Vizion digital viewing system), after 7, 10, 14, and 21 days of incubation. In addition, all EQA strains were subjected to whole-genome sequencing and phenotypically characterized by the 7H10/7H11 agar proportion method (APM) and by use of MGIT960 mycobacterial growth indicator tubes. We concluded that the UKMYC5 plate is optimally read using the Vizion system after 14 days of incubation, achieving an interreader agreement of 97.9% and intra- and interlaboratory reproducibility rates of 95.6% and 93.1%, respectively. The mirrored box had a similar reproducibility. Strains classified as resistant by APM, MGIT960, or the presence of mutations known to confer resistance consistently showed elevated MICs compared to those for strains classified as susceptible. Finally, the UKMYC5 plate records intermediate MICs for one strain for which the APM measured MICs close to the applied critical concentration, providing early evidence that the UKMYC5 plate can quantitatively measure the magnitude of resistance to anti-TB compounds that is due to specific genetic variation.
Highlights
The UKMYC5 plate is a 96-well microtiter plate designed by the CRyPTIC Consortium (Comprehensive Resistance Prediction for Tuberculosis: an International Consortium) to enable the measurement of MICs of 14 different antituberculosis compounds for Ͼ30,000 clinical Mycobacterium tuberculosis isolates
The UKMYC5 plate was designed by the CRyPTIC Consortium
The laboratory scientists were unable to read as many plates by using a mirrored box, this reading method achieved levels of reproducibility indistinguishable from those for the Vizion system and is less expensive so may be the preferred solution in low-income settings
Summary
The UKMYC5 plate is a 96-well microtiter plate designed by the CRyPTIC Consortium (Comprehensive Resistance Prediction for Tuberculosis: an International Consortium) to enable the measurement of MICs of 14 different antituberculosis (anti-TB) compounds for Ͼ30,000 clinical Mycobacterium tuberculosis isolates. Neither clinical breakpoints nor epidemiological cutoffs have been defined for any microtiter plate, nor have any plate-based assays included both new and repurposed drugs that will be key to the successful treatment of individual MDR-TB cases in the future. We designed the UKMYC5 plate, which is a variant of the MYCOTB plate, to enable the CRyPTIC Consortium (Comprehensive Resistance Prediction for Tuberculosis: an International Consortium) to measure the MICs of 14 different anti-TB compounds (see Fig. S1 and Table S1 in the supplemental material) for a large number (Ͼ30,000) of clinical TB isolates that are being collected globally by participating laboratories between 2017 and 2020. The MICs need to be quantitatively reproducible and accurate
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